Cancer Letters xxx (2014) xxxxxx

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Cancer Letters
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Mini-review

Acetate/acetyl-CoA metabolism associated with cancer fatty acid synthesis: Overview and application
Yukie Yoshii , Takako Furukawa, Tsuneo Saga, Yasuhisa Fujibayashi
Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan

a r t i c l e

i n f o

a b s t r a c t
Understanding cancer-specic metabolism is important for identifying novel targets for cancer diagnosis and therapy. Induced acetate/acetyl CoA metabolism is a notable feature that is related to fatty acid synthesis supporting tumor growth. In this review, we focused on the recent ndings related to cancer acetate/acetyl CoA metabolism. We also introduce [1-11C]acetate positron emission tomography (PET), which is a useful tool to visualize up-regulation of acetate/acetyl CoA metabolism in cancer, and discuss the utility of [1-11C]acetate PET in cancer diagnosis and its application to personalized medicine. 2014 Elsevier Ireland Ltd. All rights reserved.

Article history: Available online xxxx Keywords: Acetate Acetyl CoA Fatty acid Positron emission tomography Metabolism

1. Introduction: Importance of fatty acid synthesis in cancer Reprogramming of metabolism is one of the hallmarks of cancer [1]. Cancer cells undergo uncontrolled rapid cell growth and typically experience a shortage of nutrition and oxygen caused by abnormal structure and function of the microvessels in solid tumors. To obtain sufcient energy and the cell components necessary for rapid proliferation, cancer cells alter their metabolism. The Warburg effect is a well-known metabolic change in cancer [2]. Activation of fatty acid synthesis is another important metabolic change in cancer. Fatty acid synthase (FASN) is a key enzyme in the synthetic pathway of fatty acids from acetyl CoA. In humans, FASN is expressed at relatively high levels in healthy liver and adipose tissue, but at low levels in other tissues [3]. In many types of human cancer, including prostate, breast, lung, ovary, bladder, stomach, oral cavity and melanoma, FASN is reported to be over-expressed [4,5] and is involved in de novo synthesis of fatty acids [6]. It is also known that the over-expression of FASN in tumors is associated with poor prognosis [4,5,7]. It has been reported that FASN produces fatty acid palmitate, which can be modied into various lipids, such as phospholipids, triglycerides, cholesterol esters, and fatty-acylated proteins, from acetyl-CoA and that the resultant lipids are used as essential constituents of cell membrane, important substrates for energy metabolism, and factors in post-translational modication and cell signaling associated with tumor progression [811]. Fatty acid synthesis by FASN has been also demonstrated
Corresponding author. Address: Molecular Imaging Center, National Institute of Radiological Sciences, Anagawa, Chiba 263-8555, Japan. Tel.: +81 43 206 3429; fax: +81 43 206 0818. E-mail address: yukiey@nirs.go.jp (Y. Yoshii).
http://dx.doi.org/10.1016/j.canlet.2014.02.019 0304-3835/ 2014 Elsevier Ireland Ltd. All rights reserved.

to play an important role in carcinogenesis by protecting cells from apoptosis [4]. Recently, we discovered that FASN inhibition not only suppressed cell proliferation but also prevented pseudopodia formation and suppressed cell adhesion, migration and invasion in prostate cancer cells [12]. In that study, we also found that FASN inhibition suppressed the genes involved in production of the intracellular second messenger arachidonic acid and androgen hormones, both of which promote tumor progression [12]. These ndings demonstrated that fatty acid synthesis mediated by FASN plays critical roles at multiple stages of tumor progression (Fig. 1).

2. Supply of acetyl CoA for fatty acids synthesis from citrate Fatty acid synthesis in cancer is carried out in the cytoplasm (Fig. 2). During this process, acetyl-CoA is the starting material. One typical pathway to supply acetyl CoA for fatty acid synthesis in cancer is regeneration of acetyl CoA from citrate, which is produced from acetyl CoA in mitochondria. Generally, glycolysis produces pyruvate, and the resultant pyruvate is transferred to the mitochondria and converted to acetyl-CoA, which enters the tricarboxylic acid (TCA) cycle. In cancer cells that have activated glycolysis over mitochondrial respiration, acetyl CoA enters a truncated TCA cycle where acetyl CoA is converted to citrate, and the resultant citrate is preferentially shuttled out of the mitochondria to cytoplasm [13,14]. The citrate transferred to the cytoplasm is converted to acetyl CoA and oxaloacetate, mediated by ATP citrate lyase (ACL). It is also demonstrated that the resultant acetyl CoA is used for de novo synthesis of fatty acids and that oxaloacetate is converted to malate and the malate is transferred to the mitochondria, where it is used to regenerate oxaloacetate, in

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Fig. 1. Functions of fatty acid synthesis mediated by FASN in cancer. FASN plays important roles in multiple stages of tumor progression.

Fig. 2. Cancer metabolic pathway. Glycolysis is a universal metabolic pathway through which glucose is broken down to pyruvate (blue arrow). Pyruvate is converted to acetyl-CoA, which is transferred to the mitochondria, via mediation of pyruvate dehydrogenase (PDH) (orange arrow) or converted to lactate in the cytosol by lactate fermentation (pink arrow). Mitochondrial citrate provided by a truncated TCA cycle in cancer cells is exported to the cytosol and converted to acetyl-CoA and oxaloacetate via ATP citrate lyase (ACL) (green arrows). Cytosolic acetyl-CoA is used for fatty acid synthesis and oxaloacetate is converted to malate and malate returns to the mitochondria, where it is converted to oxaloacetate in cancer cells. Cytosolic acetyl-CoA synthetase (ACSS2) mediates interconversion between acetyl-CoA and acetate in order to maintain metabolic balance in cancer cells (red arrows). Acetyl-CoA is used for fatty acid synthesis via fatty acid synthase (FASN). In cancer cells, glycolysis, fatty acid synthesis via ACL, ACSS2, and FASN, and transfer of oxaloacetate to mitochondria is specically elevated. Respiration deciency or hypoxia causes activation of glycolysis, ACL, ACSS2, and FASN (see text for details).

cancer cells [14,15]. Thus, ACL produces cytosolic acetyl-CoA pool as a raw material for fatty acid synthesis and this enzyme therefore plays an important role in cancer cell survival.

3. Supply of acetyl CoA for fatty acids synthesis via acetate/ acetyl-CoA metabolism with acetyl-CoA synthetase (ACSS) Another pathway to supply acetyl CoA for fatty acid synthesis in cancer is a conversion of acetyl-CoA from acetate with acetyl-CoA synthetase (ACSS). ACSS (EC 6.2.1.1: ATP + acetate + CoA M AMP + diphosphate + acetyl-CoA) is recognized to play an important role in acetate/acetyl-CoA metabolism in cancer cells [16,17]. In

mammalian cells, there are two types of ACSS: mitochondrial ACSS1 and cytosolic ACSS2 [18]. We found that the expression of cytosolic ACSS2 is up-regulated in four cancer cell lines (lung carcinoma, melanoma, colon carcinoma, and mammary carcinoma), while the expression of ACSS1 is low [16]. Yun et al. examined two hepatocellular carcinoma cell lines and found that both cell lines strongly expressed ACSS2, but expression of ACSS1 varied [17]. ACSSs are generally perceived to play a role in the incorporation of acetate into acetyl-CoA, via the forward reaction [1821]. In fact, in cancer cells, [1-14C]acetate uptake is elevated when compared to normal cells, and ACSS2 is involved in the incorporation of [1-14C]acetate into acetyl-CoA [16]. The metabolic fate of radiolabeled acetate taken up by cancer cells has been well studied.

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Fig. 3. Uptake of radiolabeled acetate in cancer. In cancer cells, radiolabeled acetate is mainly used for fatty acid synthesis rather than breakdown to CO2 via tricarboxylic acid cycle or amino acid synthesis (left). In the presence of FASN inhibitors, acetate uptake and the incorporation into fatty acids are inhibited (right).

Yoshimoto et al. demonstrated that radiolabeled acetate taken up by cancer cells is mainly used for fatty acid synthesis rather than being broken down to CO2 via the TCA cycle or used for amino acid synthesis [22]. Salem et al. demonstrated that phosphatidylcholine and triacylglycerol are the predominant metabolites retained in the cells from the radiolabeled acetate in hepatocellular carcinoma in vitro and in vivo [23]. In addition, it has been reported that, in the presence of FASN inhibitors, uptake of radiolabeled acetate and the incorporation into fatty acids are reduced in prostate cancer cells [10,24]. Thus, acetate uptake mediated by ACSS2 is associated with de novo fatty acid synthesis in cancer cells (Fig. 3). Furthermore, this fatty acid synthesis with acetyl CoA produced from acetate is considered to be useful in cancer cells, especially under conditions of low cellular glucose uptake where the regeneration of acetyl CoA from citrate is limited [14].

5. Buffering role of ACSS2 in acetate/acetyl-CoA metabolism As mentioned above, ACSSs are generally recognized to play a role in the incorporation of acetate into acetyl-CoA, via the forward reaction [1821]. Interestingly, our recent data demonstrated that cancer ACSS2 also shows the reverse reaction from acetyl-CoA to acetate, i.e., acetate production from acetyl-CoA, in in vitro studies with multiple cancer cell lines [27]. In the study, we observed that cancer cells produced acetate via ACSS2 and released it into culture media and that the quantity increased under hypoxia, where the intracellular acetyl-CoA levels are high [27]. In these experiments, since we conrmed that acetate was not contained in the original medium to culture the cells, the acetate levels in the surrounding environment of the cells are quite low and thus the shifted equilibrium could have promoted the reverse reaction from acetyl-CoA to acetate mediated by ACSS2. Anyhow, ACSS2 is a bi-directional enzyme to mediate the reversible reaction between acetyl-CoA and acetate in cancer cells and could play a buffering role in acetate/ acetyl-CoA metabolism as the basis of fatty acid synthesis in the cytosol of cancer cells (Fig. 2). In addition, our data showed that expression of ACSS2 is up-regulated under hypoxia and that ACSS2 inhibition in cancer cells enhanced cell death under long-term hypoxia in vitro [27]. This indicates that ACSS2 may play an important role in the survival of cancer cells under hypoxia by not only providing acetyl-CoA through the forward reaction, but also maintaining the pool of acetyl-CoA by reducing excess acetyl-CoA through the reverse reaction in cancer cells. ACSSs are known to be well-conserved enzymes and universally distributed from bacteria to humans [28]. It has also been demonstrated that the activity of ACSSs can be controlled by acetylation of lysine residues and that this regulatory mechanism is conserved from bacteria to humans [29,30]. Previously, the enzymatic reversibility of cytosolic ACSS has been reported from several sources, including mammals, yeast, plants and bacteria [3135]. With the cytosolic ACSS of yeast and a fungus Aspergillus nidulans, the reverse reaction from acetyl-CoA to acetate is conducted under conditions of insufcient oxygen supply and would play an important role in their anaerobic survival [3641]; the reverse reaction of cytosolic ACSS contributes to reducing intracellular acetyl-CoA concentrations and generating ATP in these organisms under hypoxia. In cancer cells, ATP generation by the reverse reaction of ACSS2 was less substantial [27], and therefore the main role of the reverse reaction of ACSS2 would be buffering the acetyl-CoA pool in cancer cells. Nonetheless, the similarity between these anaerobic organisms and cancer cells, with regard to the presence

4. Fatty acid synthesis and acetate incorporation in cancer under hypoxia Up-regulation of fatty acid synthesis under hypoxia has also been reported in cancer. Furuta et al. showed that hypoxia up-regulates FASN via phosphorylation of Akt followed by activation of hypoxia-inducible factor 1 (HIF-1) in various types of cancer cells [25]. In addition, the authors demonstrated that expression of FASN is activated by H2O2 generation under hypoxia [25]. Also, the authors demonstrated that fatty acid synthesis mediated by FASN has anti-apoptotic effects in tumor under hypoxic conditions, which are normally proapoptotic, and that this function of FASN induces chemoresistance under hypoxia. It is also considered that fatty acid synthesis mediated by FASN has a role to rebalance redox by consuming NADPH, which is increased under hypoxia [25]. These evidences suggest that activation of fatty acid synthesis via FASN plays important roles in tumor survival under hypoxia. Lfer and Schneider reported that the proportion of [U-14C]acetate incorporated into lipids is elevated under hypoxia in Ehrlich ascites tumor cells and that the acetate incorporation into the lipid-soluble fraction tended to be enhanced in tumor cells under hypoxia [26]. We demonstrated that [1-14C]acetate uptake is elevated under hypoxia in four cancer cell lines, and this increase is mediated by ACSS2, the expression of which is higher under hypoxia [27]. Taken together, these results suggest that the uptake of acetate for fatty acid synthesis in cancer cells is enhanced under hypoxia. In other words, radiolabeled acetate would be a useful marker that shows the activated fatty acid synthesis under hypoxia.

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of the reverse reaction of ACSS, is useful for understanding the nature of cancer cells. 6. Clinical application of [1-11C]acetate PET for cancer Cancer cells exhibited activated acetate/acetyl-CoA metabolism mediated via ACSS2, which would contribute to providing the acetyl-CoA pool required for increased fatty acid synthesis (Fig. 3). Diagnosis of cancer by [1-11C]acetate PET is based on this mechanism. [1-11C]acetate PET is able to detect cancer by exogenously administering [1-11C]acetate and detecting the incorporated acetate in fatty acids by PET. [1-11C]acetate is transported across the cell membrane by proton-coupled monocarboxylate transporters, which are abundantly expressed and widely distributed in cells [42]. In cancer cells, transported [1-11C]acetate is actively converted to acetyl-CoA and trapped in fatty acids, compared to normal cells [1623]. It is therefore considered that the cancer-specic fatty acid synthesis from [1-11C]acetate, rather than the transporters, is critical in [1-11C]acetate PET for cancer. [1-11C]acetate PET is now clinically used for many various types of cancer, e.g., prostate cancer [43,44], renal carcinoma [45], hepatocellular carcinoma [46], brain astrocytoma [47] and glioma [48], and its signicance has been recognized in the eld of nuclear medicine. Originally, [1-11C]acetate PET was used for the evaluation of myocardial blood ow and oxidative metabolism [4952]. In the myocardium, the uptake mechanisms of radiolabeled acetate are well-understood, i.e., ACSS1 distributed in the mitochondria contributes to the uptake of radiolabeled acetate [18,31,53]. The radiolabeled acetate taken up by the heart is mainly oxidized into CO2 via the TCA cycle, and only partly used for fatty acid synthesis [18,5457]. Therefore, [1-11C]acetate PET is employed to evaluate oxygen metabolism in cardiology. In contrast, cancer cells mainly use the taken up acetate for fatty acid synthesis, as noted above. Thus, the main metabolic pathway on which [1-11C]acetate PET is based differs between the heart and cancer. 7. [1-11C]acetate PET as a predictor of FASN-targeted therapy outcome We recently demonstrated the potential of [1-11C]acetate PET as a predictor of FASN-targeted therapy outcome [12]. As noted

above, over-expression of FASN in tumors is associated with poor prognosis [4,5,7] and FASN plays an essential role at multiple stages of tumor progression [4,810,12]. Inhibitors of FASN have been reported to have anti-cancer activity and various inhibitors are now being developed [58]. Orlistat, a selective inhibitor of FASN, is one of those poised for clinical use. This is because orlistat is already used as an over-the-counter drug for obesity in the United States and European Union and the anti-cancer activity of this drug has been pre-clinically demonstrated [5,10]. FASN-targeted therapy with FASN inhibitors is therefore expected to be useful clinically; however, based on pathological studies, large variations in FASN expression levels in individual tumors have been reported [4,7]. For example, Rossi et al. examined FASN expression levels in primary prostate cancers from 64 patients and reported that FASN expression was strong in 8%, moderate in 30% and weak in 53%, with 9% showing no expression [7]. Therefore, considering the clinical application of FASN-targeted therapy, methods to predict therapeutic outcome in individual tumors before treatment are necessary in order to reduce unnecessary treatment and needless costs for patients. Va vere et al. have demonstrated that radiolabeled acetate uptake is correlated with FASN expression and [1-11C]acetate PET is a useful tool to examine FASN expression levels in xenograft of prostate cancer cell lines [24]. We recently demonstrated that tumor uptake of radiolabeled acetate reects FASN expression levels and sensitivity to FASN-targeted therapy using prostate cancer cell lines in vitro and in vivo [12]. This means that [1-11C]acetate PET is a powerful tool for accomplishing personalized FASN-targeted therapy via non-invasive visualization of tumor acetate uptake and selection of responsive tumors before treatment (Fig. 4). 8. Other probes and their possibility An analog of [1-11C]acetate, [18F]uoroacetate, is being developed [59,60] and has been shown to be useful for measurement of glial metabolism and detecting prostate cancer in PET studies [6163]. This probe could have practical advantages because of the labeling with 18F, which has a longer half-life of 2 h, when compared to 20 min for [11C]. On the other hand, it has been reported that the metabolic fate is different between [1-11C]acetate and [18F]uoroacetate in glial metabolism; [1-11C]acetate is

Fig. 4. Potential application of [1-11C]acetate PET. Scheme showing [1-11C]acetate PET as a predictor of FASN-targeted therapy outcome (A). PET imaging with [1-11C]acetate in tumor xenograft-bearing mice (B). LNCaP tumor with high FASN expression and high uptake of [1-11C]acetate showed high sensitivity to FASN-targeted therapy (left), while DU145 tumor with low FASN expression and low uptake of [1-11C]acetate showed less sensitivity to FASN-targeted therapy (right). Yellow arrows indicate tumors.

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metabolized to CO2 via TCA cycle, while [18F]uoroacetate is metabolized to uoroacetyl-CoA and then uorocitrate, which cannot be further metabolized [63]. Therefore, the difference between these two probes in metabolism should be taken into account for interpretation of the PET data. Hyperpolarized [1-13C]acetate is also being investigated as a tracer to monitor acetate ux with magnetic resonance (MR) [64]. It has been reported that, in mice, some metabolites, such as acetyl CoA and acetyl carnitine, can be detected in muscle, liver, and heart after injection of hyperpolarized [1-13C]acetate [65,66]. Since the effect of hyperpolarization is short-lived (a few tens of seconds for 13C), the measurement time in this method is limited. However, the hyperpolarized [1-13C]acetate has an advantage to provide a radiation-free method. Therefore, the application of this method to cancer diseases would have potential. 9. Conclusions Induced acetate/acetyl CoA metabolism is an important cancerspecic feature and is related to the fatty acid synthesis that is essential for tumor growth. [1-11C]acetate PET is a powerful tool for visualizing up-regulation of acetate/acetyl CoA metabolism in cancer through exogenous administration of [1-11C]acetate and detection of incorporated acetate. [1-11C]acetate PET is useful in both cancer diagnosis and predicting FASN-targeted therapy outcome, which has potential in clinical applications and developing cancer treatment. Conict of Interest The authors disclose no potential conicts of interest. Acknowledgements Original work conducted by Y.Y. was supported by JSPS KAKENHI Grant Numbers 19790864 and 23791403. We are grateful to the staff of the Molecular Imaging Center, National Institute of Radiological Sciences, Japan and the Biomedical Imaging Research Centre, University of Fukui, Japan. References
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